【摘要】：高等植物主要通過絡合作用和區(qū)室化作用進行重金屬解毒,而谷胱甘肽合酶和植物絡合素合酶是植物體重金屬解毒途徑的關(guān)鍵酶。本研究以煙草(Nicotiana tabacum)為材料,克隆煙草谷胱甘肽合酶基因(NtGSH2)和植物絡合素合酶基因(NtPCS1)。通過序列比對,組織表達模式,重金屬誘導表達等方面分析NtGSH2和NtPCS1基因的基本分子特征。并通過異源表達和過量表達分析NtPCS1基因的生物學功能。結(jié)果顯示:(1)來自不同物種的GSH2蛋白的氨基酸序列高度保守,而PCS蛋白的N端結(jié)構(gòu)域序列高度保守。(2)NtGSH2和NtPCS1基因在煙草的根、莖、葉、花和種子均表達,且均在花器官中的表達量最高。(3)NtGSH2和NtPCS1基因的轉(zhuǎn)錄表達均受到Cd~(2+)和Zn~(2+)的誘導而上調(diào)。(4)NtPCS1基因編碼植物絡合素合酶蛋白,且Ag~(2+)和Cd~(2+)能夠顯著增加植物絡合素合酶蛋白的活性。(5)NtPCS1基因的異源表達能夠增強擬南芥Atpcs1突變體對Cd~(2+)的耐受性(6)NtPCS1基因的過量表達導致煙草對Cd~(2+)超敏感。(7)NtPCS1基因的過量表達導致煙草體內(nèi)PCs含量的顯著增加。(8)外源GSH的添加能夠減弱過表達轉(zhuǎn)基因煙草對Cd~(2+)的超敏感。(9)外源GSH的添加導致過表達轉(zhuǎn)基因煙草體內(nèi)積累更多的Cd~(2+)和Zn~(2+)。
[Abstract]:High plants detoxify heavy metals mainly by complexation and compartmentalization, and glutathione synthase and plant complexin synthase are the key enzymes in detoxification pathway of heavy metals in plants. In this study, tobacco glutathione synthase (NtGSH2) gene and plant complex synthase gene (NtPCS1) were cloned from tobacco (Nicotiana tabacum). The basic molecular characteristics of NtGSH2 and NtPCS1 genes were analyzed by sequence alignment, tissue expression patterns and heavy metal induced expression. The biological function of NtPCS1 gene was analyzed by heterologous expression and overexpression. The results showed that: (1) the amino acid sequence of GSH2 protein from different species was highly conserved, while the N-terminal domain of PCS protein was highly conserved. (2) NtGSH2 and NtPCS1 genes were expressed in roots, stems, leaves, flowers and seeds of tobacco. (3) the transcription expression of NtGSH2 and NtPCS1 genes were up-regulated by Cd~ (2) and Zn~ (2). (4) NtPCS1 gene encodes plant complex synthase protein. Ag~ (2) and Cd~ (2) could significantly increase the activity of plant complexin synthase protein. (5) the heterologous expression of NtPCS1 gene could enhance the tolerance of Arabidopsis Atpcs1 mutants to Cd~ (2). (6) the overexpression of NtPCS1 gene resulted in tobacco being hypersensitive to Cd~ (2) NtPCS1 gene. (8) the addition of exogenous GSH could weaken the hypersensitivity of overexpression transgenic tobacco to Cd~ (2). (9) the addition of exogenous GSH resulted in the accumulation of more Cd~ (2) and Zn~ (2) in over-expressed transgenic tobacco.
【學位授予單位】：西南科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2

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